Perchlorate-Reducing Bacteria from Hypersaline Soils of the Colombian Caribbean

Perchlorate (ClO4−) has several industrial applications and is frequently detected in environmental matrices at relevant concentrations to human health. Currently, perchlorate-degrading bacteria are promising strategies for bioremediation in polluted sites. The aim of this study was to isolate and characterize halophilic bacteria with the potential for perchlorate reduction. Ten bacterial strains were isolated from soils of Galerazamba-Bolivar, Manaure-Guajira, and Salamanca Island-Magdalena, Colombia. Isolates grew at concentrations up to 30% sodium chloride. The isolates tolerated pH variations ranging from 6.5 to 12.0 and perchlorate concentrations up to 10000 mg/L. Perchlorate was degraded by these bacteria on percentages between 25 and 10. 16S rRNA gene sequence analysis indicated that the strains were phylogenetically related to Vibrio, Bacillus, Salinovibrio, Staphylococcus, and Nesiotobacter genera. In conclusion, halophilic-isolated bacteria from hypersaline soils of the Colombian Caribbean are promising resources for the bioremediation of perchlorate contamination.


Introduction
Perchlorate pollution is a problem of global impact because it has a negative effect on ecosystems with a loss of environmental quality, which increases with anthropogenic activity. Perchlorate is an ubiquitous emerging contaminant produced from both natural and anthropogenic sources [1], particularly present in areas associated with the use and manufacture of rockets and ammunition. is compound is a potent endocrine disruptor that mainly affects the fixation of iodine by the thyroid gland, responsible for regulating metabolism, growth, and development [2,3], thus being dangerous to infants and young children [4]. Acute, shortterm exposure has been shown to affect the nervous, respiratory, immune, and reproductive systems [5]. It has also been related to thyroid cancer and teratogenesis during the first trimester of pregnancy [6].
Perchlorate is highly distributed in ecosystems and organisms; thus, it is frequently found in several matrices, including breast milk, fertilizers, plants, food, and human tissues. is scenario has led to the prioritization of studies that allows for the removal of this contaminant from polluted sites, as it is extremely toxic and persistent; therefore, efficient treatments for its degradation are needed in order to maintain the quality of soils from biodiversity hotspots.
ere are different physicochemical techniques commonly used for the environmental removal of this anion, such as ion exchange, but it is not selective and the process usually only separates the perchlorate from contaminated sources [7], also generating by-products, which requires subsequent treatment [7]. Perchlorate is persistent but possesses biodegradability [8]. However, enzymes such as the perchlorate reductase and superoxide chlorite carry out the reduction or elimination of perchlorate. A reductase enzyme is responsible for reducing perchlorate to chlorate and chlorate to chlorite, while the enzyme superoxide chlorite changes chlorite to chloride and molecular oxygen. Biological reduction through the use of bacteria completely degrades the perchlorate ions into Cl − and O 2 (equation (1)) [9][10][11]. e perchlorate degradation pathway is as follows: Marine soils usually contain bacterial species with biochemical versatility and ability to tolerate salt, being an interesting target for researchers due to the potential reduction of environmental perchlorate [10]. e reason for selecting this type of environment is that degradation of perchlorate may be carried out using salt-tolerant bacteria [12], although this perchlorate-reduction process could be impaired with increasing salinity [11,13]. Moreover, these organisms are available in diverse environments, from Antarctica, saline lakes, and hot springs, even in hyperthermophilic and hypersaline soils [10,11]. Additionally, perchlorate deposits in these environments may be formed by chemical reactions between sodium chloride from land or sea and ozone [14].
Pilot testing of biotechnologies using perchloratereducing bacteria has been studied and tuned in suspension, fixed-bed, fluidized, and biofilm reactors [15,16], evaluating the effectiveness of treatments on soil and water contaminated with perchlorate [15]. Nowadays, the use of integrated systems, combining physicochemical treatments and perchlorate-reducing halophilic bacteria, is being studied to increase the efficiency of water treatment, allowing the handling of residual flows with high salinity and large perchlorate concentrations, simultaneously solving the waste disposal problem [17].
Colombia has a variety of ecosystems with different climatic and biogeochemical features, which facilitates the development of different halophilic bacteria. e aim of this study was the characterization of perchlorate-reducing bacteria recovered from hypersaline soils of the Colombian Caribbean.

Isolation of Strains and Culture
Conditions. Isolation, purification, and preservation techniques were used as described by Shimkets and Rafiee [19]. Samples were treated with amphotericin B (0.25 mg/mL) for 3 h until inoculation (50 µL) in the isolation media. Subsequently, samples were incubated in Petri dishes containing modified sterilized agar Luria-Bertani in seawater (LB NaCl) [20] and incubated at 37°C for 24 h under aerobic conditions [21]. Bacterial growth was monitored by observation of colonies. For preservation, a bacterial colony was transferred to modified LB broth and incubated at 37°C aerobically during 12 h, adjusting the cell density to 0.5 Mc-Farland turbidity standard. en, 720 µL of each culture was transferred to a Cryovial with 80 µL glycerol and stored at −80°C.

16S rRNA Gene Sequencing and Phylogenetic Analysis.
For 16S rRNA gene amplification, genomic DNA was extracted using a QIAamp ® DNA Mini Kit (Qiagen, CA, USA) as described by the manufacturer. e 16S rRNA gene was amplified from the total genomic DNA of the bacterial strains by PCR using the forward primer PF (5′-AGAGTTTGATCCTGGCTCAG-3′) and the reverse primer 1492R (5′-ACCTTGTTACGACTT-3′) [22,23]. PCR was performed with an AmpliTaq Gold ® 360 Master Mix (Applied Biosystems) according to the manufacturer's instructions. Each 25 μL reaction mixture contained 0.4 μM of each primer and ∼100 ng of template DNA. e amplification was performed as follows: initial denaturation for 10 min at 95°C, 25 cycles of denaturation for 1 min at 94°C, annealing for 1 min at 43°C, and extension for 1.5 min at 72°C, and a final extension for 5.5 min at 72°C. All PCR products were checked by electrophoresis on 1.2% (w/v) agarose gels stained with ethidium bromide (10 mg/mL) and analyzed using a gel documentation system (IngGenius 3 System-Syngene). e PCR product was purified with a QIAquick PCR purification kit (Qiagen, CA, USA), following the manufacturer's instructions. Automated DNA sequencing was performed by the National Center for Genomic Sequencing (CNSG) (Medellin-Colombia) using PF and 1492R primers. Sequence readings were edited and assembled with the CAP3 software [24]. e resulting 16S rRNA gene sequences were compared to reference strains with validly published names using the EzTaxon-e server (http://www. ezbiocloud.net/eztaxon). After multiple alignments of the data via CLUSTAL_W, four methods, including neighbor joining (NJ) [25], maximum likelihood (ML) [26], minimum evolution (ME), and maximum parsimony (MP) [27], were used to perform phylogenetic analysis. Phylogenetic trees were constructed using MEGA version 6 [28]. Distances were calculated using the Kimura correction in a pair-wise deletion manner [29]. e topologies of the phylogenetic tree were evaluated by the bootstrap resampling method described by Felsenstein [30] with 1000 replicates.

Morphological Characterization.
Strains were incubated on LB NaCl agar, and both growth and morphogenesis were observed under light microscopic examination (Olympus microscope BX41). Gram staining was conducted following Bergey's Manual Taxonomic Key [31] and Koneman's Microbiologic Atlas [32]. Samples were further processed by means of scanning electron microscopy (SEM) visualization as previously described [33].

Biochemical Characterization.
Biochemical characteristics were investigated using the BBL Crystal ™ Kit (Becton Dickinson Microbiology Systems, Cockeysville, USA) as described by the manufacturer. Catalase and oxidase tests were performed according to the reported methods [21].
2.6. Chloride Susceptibility Assay. All strains were assayed for perchlorate susceptibility in LB broth in the presence of NaCl (3.5, 5.0, 7.5, and 30% w/v) [20,34]. e experiments started adding 20 µL of cell suspension, OD � 0.6, into 5 mL LB broth. e cultures were incubated at 37°C for 24 h, and after that, strains showing turbidity [35] were identified as resistant to NaCl. e experiments were carried out by triplicates and performed three times.
e experiments were carried out as described for the chloride susceptibility assay. After the incubation time (24 h), a culture of each isolate was tested on LB agar at their corresponding KClO 4 concentrations to confirm cell viability and purity of each of the bacterial strains.

Evaluation of Perchlorate Reduction by Isolates.
e experiments were carried out using concentration of 10000 mg/L KClO 4 in LB with 3.5% NaCl, inoculating with the isolates as described in the chloride susceptibility assay and incubating for a 24-hour time period at 37°C. After incubation time, the final KClO 4 concentration was measured with a ermo Scientific Orion 93 perchlorate electrode ( ermo Fisher Scientific Inc., Beverly, MA), used according to the manufacturer's instructions. e difference in concentration after and before the incubation was employed to calculate the perchlorate reduction percentage elicited by each strain.   Levels of 16S rRNA gene sequence similarity between strains BBCOL-024 and BBCOL-028 and other current members of the genus Bacillus were below 99.0%. In the neighbor-joining ( Figure 2) and the minimum-evolution phylogenetic dendrograms based on 16S rRNA gene sequences, strains BBCOL-024 and BBCOL-028 were placed in a cluster in Bacillus and were shown to be closely related to B. vallismortis, B. subtilis subsp. spizizenii TU-B-10, B. vanillea, and B. atrophaeus.
For strain BBCOL-023, 1329 nt of the 16S rRNA gene sequence was determined. Comparative 16S rRNA gene sequence analysis showed that strain BBCOL-023 was most closely related to members of the genus Nesiotobacter. Strain BBCOL-023 shares the highest sequence similarity with Nesiotobacter exalbescens LA33B (T), Roseibium aquae DSG-S4-2 (T), and Pseudovibrio hongkongensis UST20140214-015B (T) with 99.8, 95.9, and 95.7% and nucleotide differences of 3, 55, and 57, respectively. e 16S rRNA gene sequence similarities to the type strains of other members of the family Rhodobacteraceae with validly published names were below 94%. In the phylogenetic tree based on the NJ algorithm (Figure 3), strain BBCOL-023 formed a single clade with Nesiotobacter exalbescens (supported by a bootstrap value of 100% (Figure 3)), with which it shares the highest 16S rRNA gene sequence similarity. e affiliation of strain BBCOL-023 and its closest neighbors was also supported by the MP and ML algorithms with above 90% bootstrap values. e 16S rDNA sequences of strains BBCOL-025, BBCOL-026, BBCOL-027, and BBCOL-031 determined in this study comprised 1402, 1361, 1399, and 1389 nt, respectively, representing approximately 90% of the Escherichia coli 16S rRNA sequence. e results of phylogenetic analysis of the 16S rRNA gene sequences revealed that the isolated strains were related phylogenetically to members of the Vibrionaceae family and belong within the phyletic group classically defined as the genus Salinivibrio and Vibrio (Figure 4). Strain BBCOL-025 shows high 16S rRNA gene sequence similarities with Salinivibrio costicola subsp. vallismortis DSM 8285(T) (98.4%), Salinivibrio costicola subsp. costicola ATCC 33508(T) (97.8%), and Salinivibrio proteolyticus AF-2004(T) (97.7%). Levels of 16S rRNA gene sequence similarity between strain BBCOL-025 and the other current members of the genus Salinivibrio are below 97%. In the neighbor joining ( Figure 4) and the minimum evolution phylogenetic dendrograms based on 16S rRNA gene sequences, strain BBCOL-025 was placed in a cluster in the Salinivibrio genus and was shown to be closely related to S. budaii and S. costicola subsp. alcaliphilus (supported by a bootstrap value of 75%). 16S rRNA gene sequence comparison between the BBCOL-026 strain and other members from the Vibrionaceae family by using the EzTaxon-e server indicated that the strain was closely related to members of Vibrio genus, showing 99.2% gene sequence similarity to V. antiquarius Ex25 (T), 99% to V. alginolyticus NBRC 15630(T), 98% to V. neocaledonicus NC470 (T), and 98.9% to V. natriegens DSM 759(T). Likewise, strains BBCOL-027 and BBCOL-031 show high 16S rRNA gene sequence similarities with V. alginolyticus NBRC 15630(T) (99.8 and 98.9%, respectively) and V. antiquarius Ex25(T) (99.5 and 98.9%, respectively). A phylogenetic tree, generated from the neighbor joining algorithm, showed that strains BBCOL-026 and BBCOL-031 both fell within the radiation of the cluster comprising Vibrio species and formed a coherent cluster that is supported by a bootstrap analysis at a confidence level of 98% (Figure 4). is cluster joins the phylogenetic clade comprising V. alginolyticus and V. parahaemolyticus, which is supported by a 71% bootstrap value.
is topology was also found in trees generated with the ML and MP algorithms (not shown). e NJ and ME phylogenetic trees based on 16S rRNA gene sequence data showed that the strain BBCOL-027 forms a coherent cluster with Vibrio alginolyticus (a bootstrap value of 72%).
For strain BBCOL-032, 1416 nt of the 16S rRNA gene sequence was determined. Comparative 16S rRNA gene sequence analysis showed that strain BBCOL-032 was more closely related to the Staphylococcus species. Strain BBCOL-032 shares highest sequence similarity with Staphylococcus haemolyticus ATCC 29970(T) (99.9%), Staphylococcus petrasii subsp. pragensis NRL/St 12/356(T), and Staphylococcus petrasii subsp. jettensis SEQ110 (T) (99.2%). e 16S rRNA gene sequence similarities to strains from other members of the Staphylococcaceae family were below 99%. In the phylogenetic tree based on the NJ algorithm ( Figure 5), strain BBCOL-032 fell within a coherent cluster comprising S. haemolyticus, S. petrasii subsp. pragensis, S. petrasii subsp. jettensis, and S. lugdunensis. e sequence similarities of strain BBCOL-032 with S. haemolyticus using different clustering algorithms (100% in NJ tree; 99% in ME tree; and 100% in ML tree) along with the EzTaxon-e server analysis consistently indicated that S. haemolyticus is the closest relative.

Microscopic and Biochemical
Characterization. e colonies of strains (BBCOL-023 to BBCOL-033) on LB agar were circular, convex, and smooth. Cells were facultative anaerobic, with an optimal growth at pH 6.5 to 7.5. Morphological features are observed by SEM (Figure 6), and biochemical characteristics of isolated bacteria strains are shown in Table 1       International Journal of Microbiology reported [38].
e strains were able to utilize lactose, mannose, galactose, sorbitol, and sucrose as carbon sources. Nitrate was reduced to nitrite. e strains were ONPG-and H 2 S-negative.
Microscopic morphological results showed that isolates BBCOL-024, BBCOL-028, BBCOL-029, and BBCOL-033 correspond to the genus Bacillus [39]. When grown at 37°C during 24 h in LB, the cells were Gram-positive bacilli, with a size between 0.6-0.7 μm and 1.6-1.7 μm. Endospores were ellipsoidal. Cells were motile, aerobic, facultative anaerobic, and oxidase-and catalase-positive. Optimal growth conditions of BBCOL-024 were at pH 6.0-6.5 and 37°C. ese isolates showed Voges-Proskauer and nitrate reduction activity. However, they were ONPG-and H 2 Snegative. e cells of strain BBCOL-025, Salinovibrio costicola, were Gram-negative, motile, nonsporulating, and curved, presenting an average bacterial size of 1.4 × 0.7 µm. In addition, these were motile, facultative anaerobic, and oxidaseand catalase-positive. Optimal growth conditions for BBCOL-025 were at pH 6.0-6.5 and 37°C. e isolates showed morphological differences regarding both curved size and growth. ese differences may arise depending on environmental conditions at the sampling moment, laboratory procedures, and conservation techniques, among others [40,41]. ese strains presented positive activity for Voges-Proskeuer and nitrate reduction and negative activity against ONPG and H 2 S production. e isolates BBCOL-026, BBCOL-027, and BBCOL-031 shared the main properties of the genus Vibrio. ey were motile, curved, facultative, Gram-negative and oxidase-positive and were able to reduce nitrate to nitrite. ese bacteria also formed yellow colonies, as reported by several authors [42,43]. Optimal growth conditions for BBCOL-025 were at pH 6.0-6.5 and 37°C. e isolates showed high phenotypical homogeneity although variable reactions were observed for aesculin hydrolysis, N-acetylglucosaminidase activity, and fermentation of galactose and lactose. e cells of strain BBCOL-032 Staphylococcus spp. were Gram-positive, motile, and nonsporulated, presenting an average bacterial size of 1.28 × 0.68 µm. Cells were facultative and oxidase-and catalase-positive. Optimal growth conditions for BBCOL-025 were at pH 6.0-6.5 and 37°C. Isolated strains presented positive activity for the Voges-Proskauer test and nitrate reduction, and showed negative activity against ONPG and H 2 S production, as previously reported [44,45].

Sodium Chloride and Perchlorate Susceptibility Assay.
All isolates showed growth in the culture medium with high concentrations of NaCl, reaching tolerance up to 30%. Strains BBCOL-023 to BBCOL-033 presented characteristics of moderately halophilic bacteria, according to what was reported by Acevedo-Barrios [20]. e ability of isolates to grow and tolerate concentrations of KClO 4 between 100 and 10000 mg/L is presented in Table 2. All isolates showed biofilm formation at the highest concentration. is barrier allows the bacteria to generate a  International Journal of Microbiology 7 concentration gradient as a means of protection against the toxicity of this chemical [2]. Other aspects evidenced in these strains are associations between NaCl and KClO 4 tolerance. ese isolates have become interesting targets for this research, given the need to identify native bacteria with potential biotechnological and biochemical versatility, capable of degrading environmental contaminants such as KClO 4 .

Evaluation of Perchlorate Reduction by Isolates.
Perchlorate-reducing bacteria are phylogenetically diverse, and these include Alphaproteobacteria, Betaproteobacteria, Gammaproteobacteria, and Deltaproteobacteria classes, with Betaproteobacteria being the most commonly detected class [46,47]. In this work, bacterial strains BBCOL-023 to BBCOL-033 (Figure 7) showed biological capacity to reduce concentrations of KClO 4 on percentages between 10 and 25. e genera Nesiotobacter and Salinivibrio showed the highest percentage (25%) of perchlorate reduction, while the genera Vibrio, Bacillus, and Staphylococcus presented a lowest proportion of KClO 4 reduction, with 14, 12, and 10%, respectively. Recent studies have shown that the amount of perchlorate reduced may be inversely proportional to increased salinity [13,17]. Future studies should be carried out to describe the role of salinity on perchlorate reduction by these strains. e ability of bacteria to grow in perchlorate polluted areas is determined by their degrading enzymes [48,49]. e general metabolic reduction pathway widely accepted by researchers [9,10] involves the reductase enzyme, as this is responsible to reduce perchlorate to chlorate and chlorate to chlorite, while the superoxide chlorite enzyme changes  International Journal of Microbiology    chlorite to chloride and molecular oxygen [9,46,50]. e optimal temperature range for perchlorate reduction is 28-37°C [46,51,52]. Perchlorate-reducing bacteria are usually anaerobic and some facultative, despite being the molecular oxygen produced as an intermediate of the microbial perchlorate reduction [46,53], in a process that exudes nitrate [46]. In our study, isolated perchlorate-reducing bacteria were also anaerobic but facultative. erefore, although these may undergo degradation processes in a wide range of environmental conditions, it is also probable that some critical anaerobic strains were missed during the aerobic treatment. In consequence, additional experiments should be carried out under anaerobic conditions, just to enrich some active microorganisms that may improve perchlorate degradation.
Perchlorate is an ubiquitous and persistent pollutant in the environment, causing toxic effects in biota and humans. erefore, different technologies have been developed to remove and eliminate this chemical. One of the most promising, effective, and economic ones is the use of bacteria in biotechnological systems that are capable of reducing and eliminating perchlorate. e rates of perchlorate reduction obtained in this study were comparable to those reported by Wang et al. [54], suggesting their potential application in bioremediation of perchlorate contaminated areas.

Conclusions
e strains isolated from Galerazamba-Bolivar, Manaure-Guajira, and Salamanca Island-Magdalena, Colombia, were halotolerant organisms belonging to the Vibrio, Bacillus, Salinovibrio, Staphylococcus, and Nesiotobacter genera. ese strains could reduce KClO 4 levels in aqueous solutions from 10 up to 25%. Bacteria-mediated remediation of perchlorate is a suitable process to control pollution by this toxic chemical.

Abbreviations
LB NaCl: Luria-Bertani in seawater ML: Maximum likelihood MP: Maximum parsimony ME: Minimum evolution NJ: Neighbor joining SEM: Scanning electron microscopy OD: Optical density.